What can be used as a loading control for western blot and why?
Beta actin.
commonly chosen as a loading control due to its general expression across all eukaryotic cell types. The expression levels of this protein do not vary drastically due to cellular treatment, which is another reason the protein makes a suitable control.
What is the inhibitor of MMP? Which types inhibit which types of MMP?
TIMP
1 - MMP2, 9, 13
2,3,4 - MT-MMP
When is MT-MMP expression the highest?
Embryogenesis ECM remodelling (wound healing, angiogenesis, cancer)
What do inactive proMMPs look like? How are they activated?
pro domain’s SH in cysteine binds with zinc in catalytic domain.
Zn2+ binding endopeptidase cleaves the pro domain
Where is proMMP cleaved?
in the pericellular environment
What are the substrate specific chacteristics in the secreted MMPs?
catalytic FN type II repeats
hemopexin domain
Characteristic of the catalytic domain in secreted MMPs?
Zn2+
FN type II repeats
FN type II is required for cleaving what?
elastins, gelatins, collagen IV,
Hemopexin is essential for cleaving what?
collagens
Whats the ratio of TIMP and MMP bind?
1:1
Explain the process of wound healing
- keratinocyte migration over basal lamina
- provisional matrix (fibrin, plasma protein) is removed
- fibroblast contracts the ECM to close the wound
Some ECM and non ECM molecules turned by MT1-MMP
ECM
- collagen I, II, III
- fibronectin
- laminin
- perlecan
- aggrecan
- syndecan 1 and 4
non ECM
- CD44
- Pro TNF alpha
Fibronect turnover is done by which MMP?
MT1-MMP
which Integrin is regulated by MT1-MMP for fibronectin turnover?
a5b1
Which molecule’s shedding activity is linked to tumor progression? and which MMP is responsible for this?
syndecans 1 and 4
MT1-MMP
Where is MT-MMP pro domain cleaved?
trans golgi
What are some differences in structure of soluble and MT MMP?
MT-MMP has
- furin recognition motif
- transmembrane domain
- Glycosyl phosphotidylinositol (GPI) anchor
Describe the activation cascade of MT1-MMP
- pro domain cleaved in trans golgi
- dimer presenting at the cell surface
- one MT1-MMP is inactivated by TIMP-2
- Other MT1-MMP cleaves pro domain in MMP2 activating it. It can also activate MMP13
- MMP2 and 13 activates MMP9
which domain allows MT1-MMP dimerization?
pex domain
What is the purpose of the furin domain?
allows trans golgi cleavage of MT1-MMP instead of extracellular
How are only half of the MT1-MMPs inactivated?
1:2 ratio of TIMP2:MT1-MMP
What do disintegrins bind do, via what?
- binds to integrins via XCD (ADAM 15 –RGD)
- also fibronectin
Venom from viper has what?
disintegrins
which ADAMs family binds to integrin a5b1?
ADAM-15 and -17
